Project Summary The germ line provides an enduring link between all generations of an organism. In mammals, the primordial germ cells (PGCs) - the founder cells of the germ line ? are specified during early post-implantation embryonic development. Understanding the development of human PGCs (hPGCs) have important implications for advancing regenerative medicine and reproductive medicine and contributing to knowledge and models of human germ cell pathologies. Despite their basic and clinical importance, the origin and specification of hPGCs remain mysterious due to interspecies divergence and limited accessibility to post-implantation human embryo samples. The primary goal of this NIH R21 project is to specifically address the great scientific challenge of studying hPGC development by exploring an in vitro, human pluripotent stem cell (hPSC)-based development model that can faithfully recapitulate post-implantation human development including hPGC specification. Specifically, in our preliminary study, we have successfully developed a hPSC-based, synthetic microfluidic embryogenesis platform in which key developmental landmarks during early human post- implantation development can be recapitulated successively in a highly controllable and scalable fashion. Excitingly, hPGCs emerge spontaneously in the synthetic embryonic tissues generated in the microfluidic platform, and they demonstrate canonical PGC markers reminiscent to those of monkey PGCs. Thus, our synthetic microfluidic platform provides a faithful and convenient embryogenesis model that opens up previously inaccessible phases of hPGC development to experimental studies. This proposed research will explore this new synthetic microfluidic human development model to study the origin and specification of hPGCs, by developing a lineage tracing system to track hPGC specification and further sorting out incipient hPGCs to examine their transcriptome dynamics during their commitment and specification. Successful accomplishment of this proposed research will lead to the establishment of an innovative microfluidics-based experimental platform with superior controllability and reproducibility for mechanistic investigations of hPGC development. By examining the temporal cascade of transcriptomic events that accompany the development of hPGCs, our research will shed light on genetic requirements and molecular mechanisms of hPGC development.